SASDB27: Chimeric EcRHH-RcPutA: The E.coli Proline utilization A RHH domai...

基本情報

登録情報

データベース: SASBDB / ID: SASDB27

試料

Chimeric EcRHH-RcPutA: The E.coli Proline utilization A RHH domain fused to R.capsulatus PutA

• Proline utilization A (protein), PutA, Escherchia coli, Rhodobacter capsulatus

• 生物種: Escherchia coli (大腸菌); Rhodobacter capsulatus (バクテリア)
• 引用: ジャーナル: Biosci Rep / 年: 2016; タイトル: Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA.; 著者: Benjamin W Arentson / Erin L Hayes / Weidong Zhu / Harkewal Singh / John J Tanner / Donald F Becker / ; 要旨: Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon-helix-helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH-RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH-RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs.

登録者

• John Tanner (Mizzou, University of Missouri-Columbia, Columbia, MO, USA)

モデル

試料

• 試料: 名称: Chimeric EcRHH-RcPutA: The E.coli Proline utilization A RHH domain fused to R.capsulatus PutA; 試料濃度: 6.00-6.00
• バッファ: 名称: 50 mM Tris, 200 mM NaCl, 0.5 mM Tris(3-hydroxypropyl)phosphine; pH: 7.5

要素 #429

名称: PutA / タイプ: protein / 記述: Proline utilization A / 分子量: 125.528 / 分子数: 2 / 由来: Escherchia coli, Rhodobacter capsulatus
配列:

MGSSHHHHHH SSGLVPRGSH MGTTTMGVKL DDATRERIKS AATRIDRTPH WLIKQAIFSY LEQLENSDTL PEHMTDLSAL GPKAKFAPEA EVLQALVAQA ALPQPQLDRI AARGADLVAR IRAEAKPSLM EHFLAQYGLS TREGVALMCL AEAMLRVPDT ATIDALIEDK IAPSDWGKHL GTAASSLVNA STWALMLTGK VLDDGAGGIA GTLRGAMRRL GEPVIRAAVG QAMREMGRQF VLGETIEKAL ERAEKREAEG YTFSYDMLGE AALTAADAER YRLAYAQAIT AIGKAATRGS IAANPGISIK LSALHPRYEV AQEARVMAEL VPVVRDLARA AARAGIALHI DAEEQDRLAL SLRVMAAVIA DPETAGWEGF GAVVQAYGKR AGAAIDALAA MARAAGRRIN IRLVKGAYWD AEMKRAQVEG HPGFPLFTSK TGTDVAYICL AAKLFGLNDC IYPQFATHNA HTVAAVLEMA AGRPFEFQRL HGMGARLHDI VLRETGGRCR IYAPVGAHRD LLAYLVRRLL ENGANSSFVN QIVNESVPPA EVAACPFAAL PTARAPRGLL APADLFGAGR VNAQGFDLSD PEVLARIEAA RDVTLPDAAP IVAGPVSGTL RPVRNPATGA VVAQVTEADA ATVALALDAA QVWSAPAATR AAVLCRAADL YEENFGPIFA ALAQEAGKTL GDAVSELREA VDFLRYYAAE GAADTRPPRG AVVAISPWNF PLAIFTGQVA AALMAGNAVL AKPAEQTPII AALAVRLLHQ AGVPETALQL LPGDGPTVGA ALTRDPRVAG VVFTGSTETA QIIARAMAAH LAPGTPLIAE TGGLNAMVVD STALPEQAVR DVVASAFRSA GQRCSALRCL YVQDDIAPHL IGMLKGAMEE LVSGDPARLS TDVGPVIDAE AKAGIETYLA ANKARILHRS TAPEGGHFVA PALLQVGGIA DLEREIFGPV LHLATFAAED LPAVIAAINA RGYGLTFGLH SRIDARVETV AETIRAGNIY VNRNQIGAVV GSQPFGGEGL SGTGPKAGGP LYLNRFYAPE PVVAVGGWTE AATPILPEAR ETQLDEIFLP GPTGELNRLT RHQRGPILCL GPGAEAASAQ AAAVVALGGQ AVQASGAVSP KALETLTPLA GVLWWGAAEM GRAYAQALAV RPGPLVPLIT AKPDLAHVAH ERHLCVDTTA AGGNAALLAG

実験情報

• ビーム: 設備名称: Advanced Light Source (ALS) 12.3.1 (SIBYLS) / 地域: Berkeley, CA / 国: USA / 線源: X-ray synchrotron / 波長: 0.1 Å / スペクトロメータ・検出器間距離: 1.5 mm
• 検出器: 名称: MAR 165 CCD

スキャン

タイトル: Chimeric EcRHH-RcPutA / 測定日: 2012年10月16日 / 保管温度: 4 °C / セル温度: 20 °C / 照射時間: 0.5 sec. / フレーム数: 2 / 単位: 1/A /

	Min	Max
Q	0.0112	0.3165

距離分布関数 P(R)

ソフトウェア P(R): GNOM 4.5a / ポイント数: 503 /

	Min	Max
Q	0.01125	0.3165
P(R) point	1	503
R	0	183

結果

カーブのタイプ: single_conc /

	実験値	Porod
分子量	251 kDa	-
体積	-	308 nm3

	P(R)	P(R) error	Guinier	Guinier error
前方散乱 I0	3033	5.5	3011.44	11
慣性半径, Rg	5.41 nm	0.008	5.2 nm	0.02

	Min	Max	Error
D	-	18.3	1
Guinier point	1	23	-